Antioxidation food product, antioxidation preparation and antioxidation method

ABSTRACT

The antioxidation food product of the present invention is a fermented product produced by adding a manganese-containing natural material such as tea and fermenting with bacteria having a catalase activity (e.g.  Lactobacillus plantarum ). The antioxidation food product has an antioxidation action in the living body including the interior of a digestive tract by expressing a superoxide dismutase-like activity and a catalase activity, simultaneously, and is effective for preventing diseases caused by active oxygen.

TECHNICAL FIELD

[0001] The present invention relates to an antioxidation food product,an antioxidation preparation and an antioxidation method, which aim atpreventing or improving diseases caused by active oxygen, by removingsuperoxide (O₂ ⁻) and hydrogen peroxide (H₂O₂).

BACKGROUND ART

[0002] Various studies on lactic acid bacteria or physiological effectsof food products using lactic acid bacteria have been made sinceMetschnikoff's longevity theory.

[0003] As a function of lactic acid bacteria contained in fermented milksuch as yogurt, for example, intestinal microflora improving effect,intestinal controlling action, etc. are well known. Recently, it hasbeen reported that lactic acid bacteria have various functions such asimmune activation action, antimicrobial action, antitumor action and thelike. As described above, since various health effects are expected forlactic acid bacteria, fermented milk and lactic acid bacteria beverageusing strains such as Lactobacillus acidophilus, Lactobacillus casei andgenus Bifidobacterium, which are also detected in the human bowel, areput on the market.

[0004] By the way, active oxygen is important as a biological protectionfactor such as bactericidal action of leucocyte, but it becomes apparentthat the excessive production of active oxygen in the living body causesvarious tissue disorders.

[0005] As the ordinary factor for generating active oxygen, there havebeen known stresses, alcohols, peroxides, drugs, exercises and the like.It is pointed out that active oxygen and lipoperoxide generated by thesefactors are closely connected with cerebral nerve diseases, circulatorydiseases, cancers, alimentary diseases, hepatic diseases, arterialsclerosis, renal diseases, diabetes, ageing and the like.

[0006] The living body retains a series of oxidation protection systemsso as to protect itself against oxygen toxicity. On the other hand, inorder to permit these systems to function normally, it is important toingest oxidation nutrient components sufficiently. As the naturaloxidation nutrient component, there have been known vitamin E, vitaminC, β-carotene, polyphenol and trace elements (e.g. selenium, copper,zinc, etc.). For the purpose of affording an antioxidation action, foodproducts containing these nutrient components have been developed.

[0007] In vivo antioxidation mechanism is classified roughly into apreventive antioxidation action (controlling the generation of aradical) and a linkage-breakage type antioxidation action (scavengingand eliminating a radical which has already been generated) according toits action. Examples of those which have the former action includeenzymes such as superoxide dismutase (SOD), catalase (CAT), glutathioneperoxidase (GSH-Px) and the like. Examples of those which has the latteraction include the above antioxidation nutrient components.

[0008] However, in a conventional food product, there have never beenknown products which aim at conducting linkage-elimination of superoxide(O₂ ⁻) and hydrogen peroxide (H₂O₂) which is connected with the lipidperoxidation.

[0009] It is an object of the present invention to provide anantioxidation food product, an antioxidation preparation and anantioxidation method, which express a superoxide dismutase (hereinafterreferred to as “SOD”)-like activity and a catalase (hereinafter referredto as “CAT”) activity, simultaneously, and which are particularlysuperior in preventive antioxidation action.

DISCLOSURE OF THE INVENTION

[0010] The antioxidation food product of the present invention has anantioxidation action in the living body including the interior of adigestive tract, comprising a fermented product produced by adding amanganese-containing natural material and fermenting with bacteriahaving a CAT activity.

[0011] That is, the present inventors have obtained a knowledge thatspecific bacteria among various lactic acid bacteria express no CATactivity under the environment in which manganese is not present, but ifmanganese is present in the growing environment, they incorporatemanganese into their cells to express a Mn-CAT activity and a SOD-likeactivity, simultaneously, and the present invention has been completed.The antioxidation food product of the present invention is particularlysuitable as a preventive antioxidation food product.

[0012] It is known that Mn-CAT is not affected by various inhibitors,modifiers, chelating agents, etc. in comparison with heme-CAT containingiron, and exhibits stability within a wide range of pH and atemperature.

[0013] Further, the antioxidation food product of the present inventionmay be a dried product, preferably freeze-dried product which containsbacteria having a CAT activity and a manganese-containing naturalmaterial, in addition to the fermented product as described above.

[0014] The present invention also provides an antioxidation preparationcomprising bacteria having a CAT activity and a manganese-containingnatural material. This antioxidation preparation may be a dried product(in the form of tablet, powder, granule, capsule, etc.) or in the formof liquid.

[0015] Furthermore, according to the present invention, there isprovided an antioxidation method in the living body including theinterior of a digestive tract, which comprises ingesting a fermentedproduct produced by adding a manganese-containing natural material andfermenting with bacteria having a CAT activity to express a SOD-likeactivity and a CAT activity, simultaneously.

BRIEF EXPLANATION OF THE INVENTION

[0016]FIG. 1 is a graph illustrating a change in SOD-like activity andCAT activity due to the addition of MnCl₂ or that of tea.

[0017]FIG. 2 is a graph illustrating a change in SOD-like activity withtime after the administration of a product produced by fermenting withLactobacillus plantarum in a pylorus-ligated rat.

[0018]FIG. 3 is a graph illustrating a change in CAT activity with timeafter the administration of a product produced by fermenting withLactobacillus plantarum in a pylorus-ligated rat.

[0019]FIG. 4 is a graph illustrating a change in viable cell number inthe pylorus-ligated stomach.

[0020]FIG. 5 is a graph illustrating a SOD-like activity before or afterthe fermentation of a tea-added product produced by fermenting withLactobacillus plantarum.

[0021]FIG. 6 is a graph illustrating an effect after the administrationof a tea-added product produced by fermenting with Lactobacillusplantarum in an indomethacin-induced gastric ulcer model.

[0022]FIG. 7 is a graph illustrating a SOD(-like) activity in serumafter the administration of a tea-added product produced by fermentingwith Lactobacillus plantarum.

[0023]FIG. 8 is a graph illustrating an effect after the administrationof a tea-added product produced by fermenting with Lactobacillusplantarum on hydrogen peroxide-induced gastric mucosal lesions.

[0024]FIG. 9 is a graph illustrating a MPO activity inhibition effect ofa tea extract solution and MnCl₂.

[0025]FIG. 10 is a graph illustrating a MPO activity inhibition effectof catechin.

BEST MODE FOR CARRYTNG OUT THE INVENTION

[0026] Examples of the bacteria having a Mn-CAT activity in the presentinvention include Lactobacillus plantarum [ATCC14431 strain] asLactobacillus.

[0027] In systematics of bacteria, it is said that lactic acid bacteriahave no CAT activity. However, when manganese is present in the growingenvironment, the above-described Lactobacillus plantarum incorporatemanganese into their cells to express a Mn-CAT activity. On the otherhand, it is known that Mn acts as a center metal of SOD and Mn itselfhas a SOD-like activity. Accordingly, it becomes possible to produce afermented product having a SOD-like activity and a CAT activity,simultaneously, by fermenting with Lactobacillus plantarum in thepresence of the manganese-containing natural material.

[0028] Recently, the clinical application of SOD against rheumatism,cardiac ischemia, etc. have been proceeded. However, there is a seriousproblem that the local amount of hydrogen peroxide is increased when SODis used alone and, further, a hydroxy radical (.OH) having a highestradical reactivity is formed. Accordingly, it is important to eliminateO₂ ⁻ and remove hydrogen peroxide by using CAT and GSH-Px in combinationwhen employing SOD. Therefore, according to the above reason, thecoupling action between SOD-like and CAT obtained by adding Mn toLactobacillus plantarum is considered to be very important.

[0029] Further, it is considered that the same effect as that ofLactobacillus plantarum can be obtained by using bacteria having a CATactivity in addition to Lactobacillus plantarum because manganese itselfhas a SOD-like activity even if no SOD is produced. Examples of thebacteria having a Mn-CAT activity other than Lactobacillus plantaruminclude Pediococcus pentosaceus, P. acidilactici, Thermolephilum albumand the like.

[0030] Further, the antioxidation food product or antioxidationpreparation of the present invention may be a dried product, preferablyfreeze-dried product comprising a bacteria having a CAT activity and amanganese-containing natural material, in addition to the abovefermented product. The dried product can be used in any form such aspowder, granule, tablet and the like. The dried product may be ingestedas it is or ingested after the dried product is added to commerciallyavailable milk and the mixture is fermented to prepare yogurt. It may bealso in the form of frozen product, in addition to the dried product.

[0031] The manganese-containing natural material in the presentinvention is used for supplying manganese to the bacteria cells. It isimpossible to add manganese as an inorganic compound because manganeseitself is not accepted as a food additive and, therefore, the naturalmaterial was used. Examples of the natural material containing a largeamount of manganese include plants such as teas, vegetables (e.g.represented by cabbage, spinach, etc.), herbs and the like. Accordingly,it is important to add these plants so as to supply manganese which isnecessary for expressing a CAT activity and a SOD-like activity tobacteria having a CAT activity. Particularly, since the tea contains alot of antioxidation components such as catechin, vitamin C, varioustrace elements, etc., high addition effect can be obtained.

[0032] Particularly, the addition of the tea to Lactobacillus plantarumcause the following effect, that is, not only the SOD-like activity andCAT activity are expressed but also the SOD-like activity is extremelyhigh in comparison with the case using an inorganic Mn compound (e.g.manganese chloride MnCl₂, etc.) and, further, the SOD-like activity andCAT activity under gastric juice exposure can be maintained for a longperiod of time.

[0033] It is preferred that the tea or the other natural material isadded in the form of powder because the incorporation of manganese dueto bacteria becomes easy. The pulverization is conducted by extracting anatural material with water and/or water-miscible organic solvent (e.g.alcohols such as ethyl alcohol, etc.) and then with an organic gosolventwhich is in-miscible to water (e.g. chloroform, ethyl acetate, butanol,etc.) to separate into an organic phase and an aqueous phase, recoveringa dissolved solid content from the aqueous phase, followed by drying.Further, a solid content may also be recovered from a solution extractedwith water and/or water-miscible organic solvent and then pulverized.Furthermore, an aqueous solution prepared by extracting the naturalmaterial with water may be added as it is without pulverizing, or thatwhich obtained by pulverizing, the natural material finely may be addedas it is.

[0034] Examples of the tea include green tea (non-fermented tea) such asrefined green tea, powdered tea, green tea of mild grade, coarse tea,dust tea, germ tea, toasted tea, etc.; fermented tea such as black tea,etc.; semi-fermented tea such as oolong tea, paochong tea (jasmine tea),etc.

[0035] In case of the fermented product, the amount of themanganese-containing natural material (amount of manganese) ispreferably about 4 to 20 mg, more preferably about 4 to 8 mg, based on 1kg of the product. In case of the dried product which mainly comprises abacteria cell, a manganese-containing natural material and an excipient,the amount of the manganese-containing natural material (amount ofmanganese) is preferably about 4 to 20 mg, more preferably about 4 to 8mg, based on 10 g of the product.

[0036] Typical examples of the form of the antioxidation food product ofthe present invention include fermented product and (freeze)driedproduct, as described above.

[0037] The fermented product in the present invention includes fermentedmilk such as yogurt and lactic acid bacteria beverage. The fermentedmilk can be obtained by adding a predetermined amount of amanganese-containing natural material into 1 liter of milk or skim milk,inoculating with lactic acid bacteria having a CAT activity andfermenting at 35 to 37° C. for about 12 to 72 hours. By adjusting theamount of the lactic acid bacteria starter and manganese-containingnatural material to be added, fermentation time, etc., there can beproduced yogurt in the form of solid (stationary yogurt), (semi)solid(stirred yogurt) and liquid (drink yogurt). Further, sweeteners (e.g.glucose, sucrose, etc.), flesh (e.g. grapefruit, apple, orange, lemon,etc.), inorganic electrolytes as a mineral source (e.g. sodium chloride,potassium chloride, magnesium chloride, calcium chloride, etc.),vitamins, flavors, etc. may be added appropriately to the fermentedmilk.

[0038] Further, the lactic acid bacteria beverage can be obtained byadding a predetermined amount of a manganese-containing natural materialto a mixed solution of skim milk and sugar (e.g. glucose, sucrose,etc.), inoculating the mixed solution with lactic acid bacteria having aCAT activity and fermenting at 35 to 37° C. for about 12 to 72 hours.Yogurt (e.g. liquid yogurt type, juice type, etc.) can be producedaccording to a proportion of skim milk to a diluting solution (e.g.water, fruit juice, etc.). As the base to be fermented, for example,there can be used milk serum, low fat milk, etc., in addition to skimmilk. Examples of the diluting solution include flesh, lactocoffee,etc., in addition to water.

[0039] The dried product in the present invention is obtained by mixingabout 5×10⁸ to 5×10¹⁰ bacteria cells with 2 to 4 g of amanganese-containing natural material, adding an excipient to themixture, followed by drying. Examples of the excipient include lactose,glucose, sucrose, origosaccharide and the like.

[0040] The dried product in the present invention may be ingested as itis or ingested after the dried product is added to milk and the mixtureis fermented at room temperature for 12 to 24 hours to prepare yogurt inthe ordinary home.

FIELD OF INDUSTRIAL APPLICABILITY

[0041] As described above, the antioxidation food product andantioxidation preparation of the present invention can express a SODactivity and a CAT activity, simultaneously, and remove superoxide andhydrogen peroxide. Therefore, they are effective for preventing diseasescaused by active oxygen.

EXAMPLES Test Example 1

[0042] (in vitro SOD-like activity and CAT activity of tea-added productproduced by fermenting with Lactobacillus plantarum)

[0043] When Lactobacillus plantarum is cultured (fermented) in an APTbroth (containing “bacto tryptone” commercially available from DIFCOLtd., yeast extract, glucose, sodium citrate, sodium chloride,dipotassium hydrogen phosphate, magnesium sulfate and sodium carbonate),MnCl₂ or tea (powdered green tea) were added in the medium so that theconcentration of Mn may be 12.5 μM, 50 μM, 100 μM and 200 μM,respectively, and the SOD activity and CAT activity of bacteria cellsand the whole medium containing MnCl₂ or tea after culturing for 16hours at the point where the multiplication of bacteria reaches themaximum were examined.

[0044] The SOD-like activity was determined by a NBT reduction methodand the CAT activity was determined by a decrease rate of hydrogenperoxide. The results are shown FIG. 1.

[0045] As shown in FIG. 1, the SOD-like activity increased in proportionto the amount of Mn to be added in both cases (addition of MnCl₂/tea),but the absolute value of the SOD-like activity in case of the additionof tea was well above (about 35 times when 200 μM of Mn is added).

[0046] The CAT activity was lower than the limit of detection when Mn isnot added. On the other hand, by adding 12.5 μM or more of Mn, anactivity of about 1800 U/200 ml of APT medium was expressed in bothcases that (addition of MnCl₂/tea).

[0047] By the way, the function of SOD and CAT is the elimination of O₂⁻ and H₂O₂. However, SOD and CAT are enzymes and, therefore, it is saidthat SOD and CAT are devitalized even if they are orally ingested and noeffect is obtained. However, since the SOD activity and CAT activity areretained in the bacteria cells in case of the Mn-added product producedby fermenting with Lactobacillus plantarum, it is expected that theseactivities are retained until the bacteria are killed. Further, asdescribed above, since Mn itself and the component in the tea have aSOD-like activity but they are not enzymes, it is considered that theyare not easily devitalized.

[0048] In order to prove the above description, the tea-added productproduced by fermenting with Lactobacillus plantarum was orallyadministered to a pylorus-ligated rat, forcibly, and a change inSOD-like activity and CAT activity with time after administration wasexamined in the following Test Examples.

Test Example 2

[0049] (Retention of SOD-like activity and CAT activity of a productproduced by fermenting with Lactobacillus plantarum in stomach ofpylorus-ligated rat)

[0050] Immediately after ligating a pylorous of SD male rats (weight:250 g), 5 ml of a product produced by fermenting with Lactobacillusplantarum was administered using an oral probe and contents in thestomach were recovered with time to examine a change in SOD-likeactivity and CAT activity. The results are shown in FIGS. 2 and 3.Further, a test product was obtained by adding MnCl₂ or a tea (powderedgreen tea) in the concentration (concentration of Mn) of 50 μM to an APTbroth and, after inoculating with Lactobacillus plantarum, fermentingfor 16 hours.

[0051] As shown in FIG. 2, in case of the addition of MnCl₂, theSOD-like activity decreased to about 35% of the initial value for 30minutes and was eliminated until 60 minutes have passed. On thecontrary, in case of the addition of tea, high activity was retaineduntil 3 hours have passed.

[0052] As shown in FIG. 3, in case of the addition of MnCl₂, the CATactivity decreased to about 80% of the initial value for 30 minutes,similar to the SOD-like activity. On the other hand, in the tea-addedgroup, the CAT activity increased by about 20% of the initial value andthe same activity as that of the initial value was expressed for 60minutes. Further, little (about 15%) activity was retained after 3hours.

[0053] As shown in FIG. 4, the viable cell number after oraladministration decreased to about {fraction (1/1000)} of the initialvalue for 60 minutes, in case of the addition of MnCl₂. After 3 hours,bacteria cells were not detected. On the other hand, in the tea-addedgroup, the viable cell number before administration was retained until60 minutes have passed. After 3 hours, bacteria cells were not detected.In FIG. 4, “ND” means that the viable cell number is lower than thelimit of detection (10⁴).

[0054] As described above, it became apparent that, by adding a tea toLactobacillus plantarum, not only the SOD-like activity and CAT activityare expressed, but also high activity is retained for a long period oftime in comparison with Mn contained in the inorganic compound such asMnCl₂. It is considered that this is caused by the retention of theviable cell number in the stomach.

Test Example 3

[0055] (SOD-like activity of water-soluble fraction of tea before andafter lactic fermentation)

[0056] As described above, high SOD-like activity can be obtained whenthe tea is added. Actually, a tea (powdered green tea) was added to aAPT broth in the concentration (concentration of Mn) of 50 μM and themedium was allowed to stand at normal temperature for 24 hours to give awater-soluble fraction (supernatant on the medium before fermentation).Then, the SOD-like activity of the water-soluble fraction was measured.As a result, as shown in FIG. 5, the SOD-like activity beforefermentation was high, 54,040 U/200 ml of APT broth.

[0057] On the other hand, the SOD-like activity of a water-solublefraction obtained by inoculating the above-described tea-added APT brothwherein Mn has been added in the concentration (concentration of Mn) of50 μM with Lactobacillus plantarum and fermenting for 16 hours wasmeasured. As a result, as shown in FIG. 5, the SOD-like activity afterfermentation was about 93,400 U/200 ml of APT broth.

[0058] As is apparent from these results, it was observed that theactivity increased by about 1.7 times that before fermentation by thefermentation. The reason is considered to be as follows. That is, theantioxidation component could be present in more effective form byutilizing the tea into Lactobacillus plantarum. From this point of view,the effect for adding the tea to Lactobacillus plantarum can beconfirmed.

Test Example 4

[0059] (Suppression of gastric mucosal lesions due to oraladministration of product produced by fermenting with Lactobacillusplantarum)

[0060] (1) Effect in indomethacin gastric ulcer model

[0061] 20 Mg/kg of indomethacin was subcutaneously administered to SDmale rats (weight: 250 g) and the effect of the product produced byfermenting with Lactobacillus plantarum to the formation of gastriculcer after 6 hours was examined. As the product produced by fermentingwith Lactobacillus plantarum, those which obtained by adding a teaextract solution (those obtained by extracting 100 g of a green tea with1 liter of hot water) to an APT broth in the concentration(concentration of Mn) of 50 μM and, after inoculating with Lactobacillusplantarum, fermenting for 16 hours.

[0062] The test product was as follows.

[0063] (a) Group of tea-added product produced by fermenting withLactobacillus plantarum: It is obtained by preparing a tea extractsolution so that its SOD-like activity may become 24,000 units/ml andadding the extract solution to a product produced by fermenting withLactobacillus plantarum. The viable cell number of Lactobacillusplantarum was 6.3×10⁹ cells/ml.

[0064] (c) Control group: only APT broth

[0065] The test product was orally administered 2 times with a dose of2.5 ml every 3 hours after indomethacin was subcutaneously administered.

[0066] The results are shown in FIG. 6. FIG. 6 is a graph illustratingan ulcer index of each group after 6 hours have passed sincesubcutaneously administration of indomethacin with the averagevalue±standard error of 10 rats. As shown in FIG. 6, the ulcer index ofthe control group was 5.8±1.8 mm. Further, it was confirmed that ulcerwas generated in all 10 rats in the control group. To the contrary, inthe group of the product produced by fermenting with Lactobacillusplantarum, the suppression was conducted significantly in comparisonwith the control group (p<0.05)

[0067] The evaluation due to the ulcer index and the measurement of theSOD-like activity which is present in a serum deproteinized componentafter the administration of the test product were conducted,simultaneously. As a result, as shown in FIG. 7, the SOD-like activityin the deproteinized component was 0.23±0.08 units/ml in the controlgroup. To the contrary, the group of the tea-added product produced byfermenting with Lactobacillus plantarum, a significant increase,2.34±0.33 units/ml (p<0.01) was exhibited.

[0068] (2) Effect in hydrogen peroxide-induced gastric mucosal lesionsmodel

[0069] In order to prepare gastric mucosal lesions due to hydrogenperoxide, SD male rats (weight: 250 g) were subjected to apylorus-ligation operation and diethylmaleic acid (0.75 ml/kg) wassubcutaneously administered as a depletion agent of glutathione in thetissue. Then, 0.5 ml of 7.5% hydrogen peroxide was orally administeredand the effect of the product produced by fermenting with Lactobacillusplantarum to the gastric mucosal lesions after 3 hours was examined.

[0070] As the product produced by fermenting with Lactobacillusplantarum, those which obtained by adding MgCl₂ or a tea extractsolution (those obtained by extracting 100 g of a green tea with 1 literof hot water) to an APT broth in the concentration (concentration of Mn)of 50 μM and, after inoculating with Lactobacillus plantarum, fermentingfor 16 hours.

[0071] The test product was as follows.

[0072] (a) Group of product produced by fermenting with Lactobacillusplantarum: It is obtained by fermenting with MnCl₂.

[0073] (b) Group of tea-added product produced by fermenting withLactobacillus plantarum: It is obtained by preparing a tea extractsolution so that its SOD-like activity may become 24,000 units/ml andadding the extract solution to a product produced by fermenting withLactobacillus plantarum. The viable cell number of Lactobacillusplantarum was 6.3×10⁹ cells/ml.

[0074] (c) Tea group: It is obtained by preparing a tea extract solutionso that its SOD-like activity may become 2,400 units/ml and adding theextract solution to an APT broth.

[0075] (d) Control group: only APT broth

[0076] The test product was orally administered with a dose of 2.5 mlimmediately before the administration of hydrogen peroxide.

[0077] The results are shown in FIG. 8. FIG. 8 is a graph illustratingan average value and a standard error of a gastric mucosal lesions index(area %) of 10 rats. As shown in FIG. 8, the erosions area was30.60±6.34% in the control group. To the contrary, in the tea group andthe group of the product produced by fermenting with Lactobacillusplantarum, the total area of the erosions was 22.38±5.59% and20.32±8.05%, respectively, and no significant difference from thecontrol group was observed. On the other hand, in the group of thetea-added product produced by fermenting with Lactobacillus plantarum,the total area of the erosions was 4.46±2.12% and exhibited a remarkablesuppression effect in comparison with the control group (p<0.05).

[0078] (3) Results

[0079] In the indomethacin-induced gastric ulcer model, the suppressioneffect of the formation of gastric ulcer due to the tea-added productproduced by fermenting with Lactobacillus plantarum was confirmed.Further, in the hydrogen peroxide-induced gastric mucosal lesions model,the effect of the administration of Lactobacillus plantarum cells or teaalone was not observed. The suppression effect was observed when theyare used in combination.

[0080] It has hitherto been explained that the formation of gastriculcer due to indomethacin is caused by deterioration of mucosalresistance and deterioration of blood flow due to the formationinhibition of prostaglandin. According to the latest report, it isconfirmed to be connected with active oxygen due to arachidonic acidmetabolic process and infiltration of neutrophil. It is considered thatit is connected with active oxygen produced in the gastric mucosalventriculi because the subcutaneous administration of indomethacin usedat this time acts on the gastric mucosal through blood from the portionadministered.

[0081] The formation of gastric ulcer was suppressed by the tea-addedLactobacillus plantarum and, at the same time, the SOD-like activity ofthe deproteinized component in the serum was increased. Therefore, it isconsidered that the antioxidation substance in the tea was absorbed toeliminate active oxygen in the gastric mucosal. It is also consideredthat examples of the gastric mucosal lesions due to the oraladministration of hydrogen peroxide include direct mucosal disorder dueto hydrogen peroxide and indirect disorder through infiltration ofneutrophil. In both cases, hydrogen peroxide which is present in thegastric lumen acts as a induced substance. The fact that the gastricmucosal lesions induced by hydrogen peroxide was suppressed byadministrating the tea and Lactobacillus plantarum cells in combinationmeans that the Mn-CAT activity of Lactobacillus planterum cells actedeffectively in the gastric lumen. On the other hand, the fact that theeffective suppression effect was not exhibited by administratingLactobacillus plantarum cells alone means that the CAT activity of thecells did not act in the gastric lumen, which reflects the results ofTest Example 2.

Test Example 5

[0082] (MPO (myeloperoxidase) activity inhibition action of tea extractand catechin)

[0083] It has been known that the neutrophil products active oxygen soas to detoxicate foreign materials and bacteria in the living body.Particularly, the neutrophil has a dimutated system of hydrogen peroxidewhich is referred to as “MPO”, thereby producing perchloric acid ormonochloramine having high cytotoxicity. At the time of the excessivereaction, the transudation of MPO to the exterior of MPO is confirmedand it is considered to be one factor to cause the disorder of thebiomembrane. There is also a report that the infiltration of neutrophilis connected with its crisis mechanism in indomethacin-induced gastriculcer and hydrogen peroxide-induced gastric mucosal lesions.

[0084] The meaning of the addition of the tea used as themanganese-containing natural material in the present invention is asdescribed above. Further, it was examined in vitro whether the teacomponent inhibits the MPO activity or not.

[0085] To 2.9 ml of a 50 mM phosphate buffer (pH: 5.4) containing 0.1%hydrogen peroxide and 1.03 mM o-dianisidine, 50 μl of a test solutionand 50 μl of a MPO purified enzyme (1.24 units/ml) were added and, afterincubating in a cuvet at 25° C., measuring a change in absorbance (λ:450 nm) was measured for 5 minutes. As a blank, 50 μl of a 50 mMphosphate buffer (pH: 5.4) was used.

[0086] As the test solution, a tea extract solution obtained byextracting a green tea with hot water and a solution obtained bymeasuring the concentration of Mn in the tea extract solution and addingMnCl₂ in an APT broth so that the concentration of Mn in the medium maybe the same as that of the tea extract solution were used. Then, the MPOactivity inhibition rate in each concentration of Mn. The results areshown in FIG. 9.

[0087] As is apparent from FIG. 9, the tea extract solution inhibits theMPO enzyme activity and the inhibition activity becomes higher as theconcentration of Mn becomes higher. Further, it exhibits high inhibitionrate in comparison with a Mn solution having the same concentration. Itis considered to be connected with the tea component other than Mn.

[0088] Therefore, it was examined whether the component contained in thetea other than Mn is connected with the MPO activity inhibition or not.That is, according to the same manner as that described above, the MPOinhibition activity was examined using a purified catechin as thecomponent other than Mn. The results are shown in FIG. 10. As isapparent from FIG. 10, catechin exhibits the inhibition effect in theconcentration of 1.5 μg/ml or more.

[0089] As described above, the inhibition of the MPO activity due to thetea component was confirmed and, at the same time, it became apparentthat Mn and catechin are connected with its activity inhibition.

Example 1 (Yogurt Type)

[0090] To 1 liter of sterilized milk, 4 g of a tea (powdered green tea)was added. Lactobacillus plantarum was inoculated into this milk whereinthe tea had been added. Then, the fermentation was conducted for 18hours while maintaining the temperature at 35 to 37° C.

[0091] The following yogurt forms could be prepared by adjusting themanufacturing conditions such as amount of the tea added, fermentationtime, etc. according to a normal method.

[0092] a. Solid (set yogurt)

[0093] b. (Semi)solid (stirred yogurt)

[0094] c. Liquid (drink yogurt)

Example 2 (Lactic Acid Bacteria Beverage Type)

[0095] A tea (powdered green tea) was added to a predetermined amount ofa mixed solution of skim milk and sucrose, and then Lactobacillusplantarum was inoculated into the mixed solution, which was fermentedfor 18 hours while maintaining the temperature at 37° C. Thereafter, thefermented solution was diluted with water and a stabilizer and a flavorwere added to give a liquid yogurt type lactic acid bacteria beverage.The composition thereof is shown in Table 1.

Example 3 (Lactic Acid Bacteria Beverage Type)

[0096] A single cell tea (manufactured by Single Cell Foods Co., Ltd.)as the tea (powdered green tea) was added to a predetermined amount of amixed solution of skim milk and sucrose, and then Lactobacillusplantarum was inoculated into the mixed solution, which was fermentedfor 18 hours while maintaining the temperature at 37° C. Thereafter, thefermented solution was diluted with water and orange juice, and astabilizer and a flavor were added to give a juice type lactic acidbacteria beverage. The composition thereof is shown in Table 1. TABLE 1Example 2 Example 3 Drink yogurt type Juice type Raw material % byweight % by weight Skim milk 40.0 5.0 Sucrose 14.0 14.0 Water 45.0 70.0Tea 0.4 0.4 Fruit juice 0.0 10.0 Others¹⁾ 0.6 0.6

Example 4 (Freeze-Dried Type)

[0097] About 5×10¹⁰ bacteria cells (Lactobacillus plantarum), 4 g of atea (powdered green tea) and an excipient such as lactose and glucosewere mixed and the mixture was freeze-dried to give a tablet typefreeze-dried product. This product was suitable for ingesting as it is.

Example 5 (Freeze-Dried Type)

[0098] About 5×10⁸ bacteria cells (Lactobacillus plantarum), 4 g of atea (powdered green tea) and an excipient such as lactose and glucosewere mixed and the mixture was freeze-dried to give a tablet typefreeze-dried product. This product was added to commercially availablemilk and the mixture was fermented at room temperature for 12 to 24hours. As a result, yogurt could be prepared. Accordingly, thisfreeze-dried product is suitable for preparing yogurt in the ordinaryhome.

1. An antioxidation food product having an antioxidation action in theliving body including the interior of a digestive tract, comprising afermented product produced by adding a manganese-containing naturalmaterial and fermenting with bacteria having a catalase activity.
 2. Theantioxidation food product according to claim 1, wherein the bacteriahaving a catalase activity are Lactobacillus plantarum.
 3. Theantioxidation food product according to claim 1, wherein themanganese-containing natural material is a tea.
 4. An antioxidation foodproduct having an antioxidation action in the living body including theinterior of a digestive tract, comprising a dried product which containsbacteria having a catalase activity, and a manganese-containing naturalmaterial.
 5. An antioxidation preparation having an antioxidation actionin the living body including the interior of a digestive tract,comprising a bacteria cell having a catalase activity, and amanganese-containing natural material.
 6. An antioxidation method in theliving body including the interior of a digestive tract, which comprisesingesting a fermented product produced by adding a manganese-containingnatural material and fermenting with bacteria having a catalase activityto express a superoxide dismutase-like activity and a catalase activity,simultaneously.